Homologous recombination is a fundamental process which is important for creating genetic diversity and for maintaining genome integrity. In E. coli RecA protein plays a central role in homologous genetic recombination in vivo and promotes homologous pairing of double-stranded DNA with single-stranded DNA or partially single-stranded DNA molecules in vitro. Radding, C. M. (1988). Homologous pairing and strand exchange promoted by Escherichia coli RecA protein. Genetic Recombination. Washington, American Society for Microbiology. 193-230; Radding, C. M. (1991). J. Biol. Chem. 266: 5355-5358; Kowalczykowski, et al., (1994). Annu. Rev. Biochem. 63: 991-1043. In the yeast Saccharomyces cerevisiae there are several genes with homology to recA gene; Rad51, Rad57 and Dmc1. Rad51 is a member of the Rad52 epistasis group, which includes Rad50, Rad51, Rad52, Rad54, Rad55 and Rad57. These genes were initially identified as being defective in the repair of damaged DNA caused by ionizing radiation and were subsequently shown to be deficient in both genetic recombination and the recombinational repair of DNA lesions. Game, J. C. (1983). Yeast Genetics: Fundamental and applied aspects. J. F. T. Spencer, D. H. Spencer and A. R. W. Smith, eds. (New-York:Springer-Verlag): 109-137; Haynes, et al., (1981). The molecular biology of the yeast Saccharomyces cerevisiae: Life cycle and inheritance. J. N. Strathern, E. W. Jones and J. M. Broach, eds. (Cold Spring harbor, N.Y.:Cold Spring Harbor laboratory press): 371-414; Resnick, M. A. (1987). Meiosis, P. B. Moens, ed. (New York: Academic Press): 157-210. During meiosis Rad51 mutants accumulate DNA double-strand breaks at recombination hot spots (Shinohara, et al., (1992). Cell 69: 457-470). Yeast rad51 gene was cloned and sequenced (Basile, et al., (1992). Mol. Cell. Biol. 12: 3235-3246; Aboussekhara, et al., (1992) Mol. Cell. Biol. 12: 3224-3234). Although yeast Rad51 gene shared homology with E. coli recA gene, the extent of homology was not very strong (27%). However, the extent of structural conservation between RecA protein and Rad51 protein became apparent when the yeast Rad51 protein was isolated and was shown to form nucleoprotein filaments that were almost identical to the nucleoprotein filaments formed by RecA protein (Ogawa, et al., (1993). CSH Symp. Quant. Biol. 58: 567-576; Ogawa, T., et al., (1993). Science 259: 1896-1899; Story, et al., (1993). Science 259: 1892-1896). Recently genes homologous to E. coli recA and yeast rad51 were isolated from all groups of eukaryotes, including mammals (Morita, et al., (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 6577-6580; Shinohara, et al., (1993). Nature Genet. 4, 239-243; Heyer, W. D. (1994). Experientia 50, 223-233; Maeshima, et al., (1995). Gene 160: 195-200). Phylogenetic analysis by Ogawa and co workers suggested the existence of two sub-families within eukaryotic RecA homologs: the Rad51-like (Rad51 of human, mouse, chicken, S. cerevisiae, S. pombe and Mei3 of Neurospora crassa) and the Dmc1-like genes (S. cerevisiae Dmc1 and Lilium longiflorum LIM15) (Ogawa, supra). All these Rad51 genes share significant homology with residues 33-240 of the E. coli RecA protein, which have been identified as a `homologous core` region.
Yeast and human Rad51 proteins have been purified and characterized biochemically. Like E. coli RecA protein, yeast and human Rad51 protein polymerizes on single-stranded DNA to form a right-handed helical nucleoprotein filament which extends DNA by 1.5 times (Story, supra; Benson, et al., (1994) EMBO J. 13, 5764-5771). Moreover like RecA protein Rad51 protein promotes homologous pairing and strand exchange in an ATP dependent reaction (Sung, P. (1994). Science 265, 1241-1243; Sung, P. and D. L. Robberson (1995). Cell 82: 453-461; Baumann, et al., (1996) Cell 87, 57-766; Gupta, et al., (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 463-468). Surprisingly, polarity of strand exchange performed by Rad51 protein is opposite to that of RecA protein (Sung and Robberson supra) and the relevance of this observation remains to be seen.
Surprisingly, studies with mouse models show that targeted disruption of the Rad51 gene leads to an embryonic lethal phenotype (Tsuzuki, et al., (1996). Proc. Natl. Acad. Sci. U.S.A. 93: 6236-6240). Moreover attempts to generate homozygous rad51-/-embryonic stem cells have not been successful. These results show that Rad51 plays an essential role in cell proliferation, a surprise in view of the viability of S. cerevisiae carrying rad51 deletions. It is also interesting to note that Rad51 was found to be associated with RNA polymerase II transcription complex (Maldonado, et al., (1996). Nature 381, 86-89), the specificity and functional nature of these interactions remains to be seen but all these observations point to a pleiotropic role of hsRad51 in DNA metabolism.
While Rad51 transcripts and protein are present in all the cell types examined thus far, the highest transcript levels are found in tissues active in recombination, including spleen, thymus, ovary and testis (Morita, supra). Rad51 is specifically induced in murine B cells cultured with lipopolysaccharide, which stimulates switch recombination and Rad51 localizes to nuclei of switching B cells (Li, et al., (1996). Proc. Natl. Acad. Sci. U.S.A. 93: 10222-10227). These findings are consistent with the view that Rad51 plays an important role in lymphoid specific recombination events such as V(D)J recombination and immunoglobulin heavy chain class switching. In spermatocytes undergoing meiosis, Rad51 is enriched in the synaptonemal complexes, which join paired homologous chromosomes (Haaf, et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2298-2302; Ashley, et al., (1995) Chromosoma 104: 19-28; Plug, et al., (1996). Proc. Natl. Acad. Sci. U.S.A. 93: 5920-5924). In cultured human cells, Rad51 protein is detected in multiple discrete foci in the nucleoplasm of a few cells by immunofluorescent antibodies. After DNA damage, the localization of Rad51 changes dramatically when multiple foci form in the nucleus and stain vividly with anti-Rad51 antibodies (Haaf, supra, 1995). After DNA damage the percentage of cells with focally concentrated Rad51 protein increases; the same cells show unscheduled DNA-repair synthesis.
Micronuclei (MN) originate from chromosomal material that is not incorporated into daughter nuclei during cell division. Different chemicals and treatment of cells induce qualitatively different types of micronuclei. MN caused by ionizing radiation or clastogens (i.e. 5-azacytidine) mostly contain acentric chromosome fragments (Verhaegen, F., and Vral, A. (1994). Radiation Res. 139, 208-213; Stopper, et al., (1995). Carcinogenesis 16, 1647-1650). In contrast, MN induced by aneuploidogens (i.e. colcemid) result from lagging whole chromosomes and stain positively for the presence of kinetochores/centromeres (Marrazini et al., 1994; Stopper, et al., (1994). Mutagenesis 9, 411-416). Determination of MN frequencies represents a good assay to measure genetic damage in cells, since it is much faster and simpler than karyotype analyses. In this light, the MN test has been widely used as a dosimeter of human exposure to radiation or clastogenic and aneugenic chemicals, and for the detection and risk assessment of environmental mutagens and carcinogens (Heddle, et al., (1991) Environmental Mol. Mutagenesis 18, 277-291; Norppa, et al., (1993). Environmental Health Perspect. 101, Supp. 3, 139-143; Hahnfeldt, et al., (1994) Radiation Res. 138, 239-245). However, although the MN assay is a convenient in situ method to monitor cytogenetic effects, the understanding of the connection between initial DNA damage and formation of MN is still poor.
The tumor suppressor p53 prevents tumor formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinational events (Lane, D. P. (1992). Nature 358: 15-16; Lane, D. P. (1993). Nature 362: 786-787; Sturzbecher, et al., (1996). EMBO J. 15: 1992-2002) reported that wild-type tumor suppressor protein p53 interacts physically with human Rad51 protein and it inhibits the biochemical functions of Rad51 like ATPase and strand exchange. In vivo temperature sensitive mutant p53 formed complexes with Rad51 only in wild type but not in mutant conformation. They suggested that gene amplifications and other types of chromosome rearrangements involved in tumour progression might occur not only as a result of inappropriate cell proliferation but as a direct consequence of a defect in p53 mediated control of homologous recombination processes due to mutations in the p53 gene. (Meyn, et al., (1994). Int. J. Radiat. Biol. 66: S141-S149) showed that normal cells transfected with a dominant-negative p53 mutant acquired interference with the G1-S cell cycle checkpoint and showed up to an 80-fold elevation in RAD51 mediated homologous DNA recombination rates compared with the normal parental control cells. Thus, loss of normal p53 function may cause a loss in control of normal DNA repair, recombination, and ultimately replication, resulting in inappropriate cell division and neoplastic growth. Breast tumour cells have mutated p53 genes and proteins and have various types of chromosomal aberrations like insertions, deletions, rearrangements, amplifications etc., indicative of abnormally controlled recombination.
Accordingly, it is an object of the invention to provides methods of diagnosis and screening which focus on Rad51.